iGEM Microbiology Intro
Prof. Sebas an Maerkl Index • Prokaryo c Structure and Func on – Cell components – Chemotaxis and flagellar – Living magnets • Nutri on and Growth – Growth requirements – Pla ng techniques – Growth curve – Chemostats / turbidostats • Microbial Gene cs – Recombina on – Transposons – Plasmids – Conjuga on – Transforma on – Transduc on Prokaryo c Cell Structure • Plasma Membrane • Gas vacuole: buoyancy • Ribosomes: protein synthesis • Inclusion Bodies: storage of C, P, etc • Nucleoid: chromosomal DNA • Periplasmic space: hydroly c and binding proteins • Cell Wall: mechanical rigidity • Capsules: resistance to phagocytosis; adhesion • Fimbriae and pili: a achments and ma ng • Flagella: movement • Endospore: survival mechanism
10.1016/j.cell.2008.11.016 Morphology and Classifica on
Gram-Positive Gram-Negative Flagella and Chemotaxis • Tumble: – CheA autophosphorylates if MCP is not bound – CheA then phosphorylates CheY, which causes a tumble (CheYp is rapidly degraded to CheY by CheZ • Run: – A ractant binds to MCP causing a stearic shi , limi ng autophosphoryla on of CheA – CheYp levels drop, causing CCW rota on • Rese ng: – CheR methylates MCPs causing increased CheA autophosphoryla on – This causes increases in CheYp and CheBp, which lead to CW rota on and de‐methyla on of MCPs Living Magnets (Magnetotac c Bacteria)
• Magnetosomes consist of magne c iron mineral crystals
made from: Fe3O4, greigite, or Fe3S4 • Magnetosomes align the bacteria in the geomagne c field, allowing for directed taxis Microbial Metabolism • Prototrophs: can thrive on minimal medium • Auxotrophs: lack the ability to synthesize a par cular organic compound (commonly used in yeast for selec on) Carbon Sources
– Autotrophs: CO2 principal carbon source – Heterotrophs: reduced, preformed organic molecules Energy Sources – Phototrophs: light – Chemotrophs: oxida on of organic or inorganic compounds Hydrogen or Electron Sources – Lithotrophs: reduced inorganic molecules – Organotrophs: organic molecules Algea: photolithotrophic autotrophs E.coli: chemoorganotrophic heterotroph Media • Synthe c or Defined Media: all components are known. Chemoorganotrophic heterotrophs can be grown on media containing glucose, ammonium salts and other salts. • Complex Media: contain undefined components such as peptones (protein hydrolysates), meat extract or yeast extract • Selec ve Media: favor par cular microorganisms • Differen al Media: permit the iden fica on of bacteria based on biological characteris cs
MacConkey agar is both selective and differential: it selects for gram- negative bacteria and stains for lactose fermentation Culturing • Genera on of Pure Cultures is extremely important in general microbiology, but also in cloning! • Two approaches: streak pla ng and spread pla ng • Spread pla ng is also used to obtain growth curves by coun ng colony forming units (CFUs) • Each colony is clonal ! They arise from a single bacterium and thus are genotypically the same. OK
BAD Spread Pla ng • Spread pla ng is also used to obtain growth curves by coun ng colony forming units (CFUs). • The most accurate count of live cells. Of course dead or dormant cells are not counted, as they do not form a colony. • Faster: op cal turbidity measurements • Bacterial lawns are generated by spread pla ng. • Transformed bacteria are generally spread plated. Batch Growth
Nt = popula on at me t
N0 = ini al popula on n = number of genera ons in me t n Nt = N0 x 2 Solve for n:
n=logNt‐logN0/log2 Mean growth rate constant (k): k=n/t Some generation times: • E.coli: 21 minutes Mean genera on me (g): • B.subtilis: 26 minutes g=1/k • S.cerevisiae: 120 minutes • M.tuberculosis: 720 minutes (12 hours) Chemostats / Turbidostats
• Chemostat: – Constant dilu on rate – D=flow rate/Volume – At steady state the specific growth rate (μ) is equal to D
– If μmax is < than D, culture will wash out • Turbidostat: – Cell density determines dilu on rate – More stable at high‐dilu on rates and low‐cell densi es
– μmax is more easily achieved Recombina on Gene c recombina on is the process by which a strand of gene c material (usually DNA; but can also be RNA) is broken and then joined to a different DNA molecule. • Prokaryo c recombina on takes place a er horizontal gene transfer (as opposed to during meiosis in eukaryotes) • Three possible ways for the horizontal transfer of DNA: • Conjuga on • Transforma on • Transduc on Transposi on
• Transposons are pieces of DNA that can “hop” in and out of target DNA sequences. • They code for a transposase which recognizes inverted repeats flanking the transposon (such a simple transposon is called inser on sequence, ISs containing other genes are called composite transposons) • Transposons play an important role in evolu on! Bacterial Plasmids • Plasmids are circular DNA molecules that can exists independently of host chromosomes and they: – Have their own replica on sites (they are a replicon). – Contain a rela vely small number of genes that are non‐ essen al to the host. – Can exists either as a single copy or mul ple copies in a cell. – Episomes are plasmid that can integrate into the host genome. – Conjuga ve plasmids carry genes coding for pili allowing them to undergo horizontal gene transfer through conjuga on. • Types of Plasmids: – Fer lity Factors: allows for conjuga on; carries genes for sex pili forma on, DNA transfer, etc. Is an episome. – R Plasmids: resistance factors; carry genes capable of neutralizing an bio cs – Col Plasmids: code for bacteriocins, which are bacterial toxins – Virulence Plasmids: make their host more pathogenic – Metabolic Plasmids: as the name implies • Vectors are plasmids used in gene c engineering. Conjuga on
• Bacterial conjuga on is the transfer of gene c material between bacteria through direct cell‐ to‐cell contact. • Lederberg and Tatum showed in 1946 that DNA could be transferred between two auxotrophic (triple auxotrophs) strains of bacteria • Bernard Davis then showed in 1950 that this DNA transfer requires direct physical contact. • F+ x F‐ conjuga on: transfers a f factor plasmid without transferring bacterial genes • Hfr conjuga on: transfer of an integrated f factor, causes transfer of chromosomal material, but as only part of the f factor is transferred recipient remains F‐ • F’ conjuga on: a Hfr host de‐integrates the f factor including part of the bacterial chromosome. The en re F’ plasmid is transferred. Transforma on
• Transforma on is the uptake of naked DNA from the environment in an inheritable form. • The natural transforma on efficiency of competent cells is on the order of 10‐3 (1 in 1000 cells takes up DNA). • Laboratory generated competent cells have efficiencies on the order of 106 – 109 transformants per μg of DNA. • Reversal of auxotrophies or an bio c resistance is o en used to select for transformants in the lab. Transduc on
• Transduc on is the process by which DNA is transferred from one bacterium to another by a virus. • Phage have two life cycles: – Lysogenic and ly c • Generalized transduc on: consequence of the ly c cycle, bacterial DNA may be randomly packaged • Specialized transduc on: if a prophage excises incorrectly and takes along a piece of chromosomal DNA Homework • Take a look at my favorite iGEM projects from 2008: – UC Berkeley: Clonebots h p://2008.igem.org/Team:UC_Berkeley – Imperial College: Biofabricator sub lis h p://2008.igem.org/Team:Imperial_College – Caltech: mul ‐func onal probio c bacteria h p://2008.igem.org/Team:Caltech – Harvard: Bactricity h p://2008.igem.org/Team:Harvard • Read up on some common/interes ng bugs: – E.coli, S.cerevisiae, H.pylori, B.sub lis, S.oneidensis, Magnetotac c bacteria Seminars
• GHI Seminar every Thursday at 12:15 in AI 1153 • Friday, March 13th 2009 ‐ 15:30pm Loca on: AI 1 153 Life without a wall or division machine in Bacillus sub lis